JP2014093972A - Artificial hatching method for sperm egg without shell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To attain sperm egg artificial hatching by a heat insulation method (lap method) while holding a sperm egg without shell in a transparent plastic film.SOLUTION: An artificial hatching method for a sperm egg without shell includes the steps of: breaking a sperm egg with shell into a recessed part of a recessed plastic film molded body without creases, formed by drawing and molding a transparent plastic film with oxygen permeability in a recessed shape, and suspended in a container; placing the container in an incubator, and retaining heat of the broken sperm egg without shell in contact with liquid water and solid calcium salt; and supplying humidified oxygen into the container for at least the last 10% period of a period from the start of the heat-retaining to hatching.

Description

  The present invention relates to an artificial hatching method for shellless sperm eggs such as birds.

  To observe the occurrence of chickens continuously, the “window opening method” in which a window is opened in the eggshell and the “tea bowl method” in which the contents are transferred to a teacup and covered with glass or the like are kept warm. In addition, the “wrap method” using a transparent plastic wrap, which is an advanced version of the teacup method, is an excellent method that allows the user to observe the desired part from all directions. However, as with the tea bowl method, normal occurrence is from the 7th day to the 9th day, and it is rare to observe until the 14th day. In addition, it has never been possible for chicks to be born for 21 days.

  According to Prof. Masamichi Uehira (Kyushu University) who succeeded in born a quail for the first time in the world using an artificial container in 1995, it is important to add calcium and improve oxygen permeability for the development of shellless eggs. It is a thing. In the experiment, the addition of calcium lactate and the white turbid Teflon (registered trademark) film having high oxygen permeability were successfully hatched. However, since 2000, research using artificial containers, including Professor Uehira, has not progressed. Prior to the present invention, there has been no report of successful hatching of shellless sperm eggs by the lapping method using a transparent plastic film.

Sakakura, K., 1984. "Learning development of chickens" Genetics. Pp.70-73. Ryuichi Takatsuki, 1987. “Research on biological teaching materials that make use of familiar nature” Toyokan Publishing Co., Ltd., National Science Education Center Research Council, pp. 180-183. M.M.Perry ・ etc, 1988. A complete culture system for the chick embryo. NATURE VOL.331 JANUARY.pp70-72 Masamichi Kamihira et al., 1995. Effects of environmental factors on extra-embryonic culture of avian embryos. The 60th Annual Meeting of the Chemical Society of Japan. Masahira Kamihira et al., 1996. Development of an avian embryo culture method in an artificial environment. The 61st Annual Meeting of the Chemical Society of Japan. Pp. 3. Masahira Kamihira et al., 1997. Development of a method for culturing avian embryos using artificial eggshell. 62nd Annual Meeting of the Chemical Society of Japan. Pp.2. Masamichi Kamihira.etc, 1998. Improved hatching for in vitro quail embryo culture using surrogate eggshell and artificial vessel.Develop Growth Differ.pp.449-445.

  An object of the present invention is to achieve artificial hatching of a sperm egg by a method of keeping a sperm without a shell in a transparent plastic film while keeping it warm (wrap method).

  As a result of repeating various trials and errors over many years, the present inventor succeeded in hatching a shellless sperm egg by a wrap method for the first time in a combination of various conditions, and completed the present invention.

That is, the present invention
A step of inserting a shelled sperm egg into a concave portion of a concave plastic film molded body without wrinkles suspended in a container, formed by stretching a transparent plastic film having oxygen permeability into a concave shape;
Placing the container in an incubator and keeping the interrupted shellless sperm egg in contact with liquid water and solid calcium salt; and
Supplying at least the last 10% of the period from the start of heat insulation to hatching, supplying humidified oxygen into the container,
Provided is a method for artificially hatching shellless sperm eggs.

  According to the present invention, a method for hatching a shellless sperm egg by the lapping method is provided for the first time. The present invention was the first in the world to succeed in hatching a shellless sperm egg by the lapping method, and as specifically described in the following examples, the hatching rate was as high as 80% (n = 5) and reproducibility. It became possible to hatch well. In the method of the present invention, since the embryo is held in the transparent plastic film until the chick is born, the state of the embryo can be visually observed from all directions. In addition, it is easy to bring various substances and genes into direct contact with the embryo, so it can be used for embryological research, drug discovery research, and the production of transgenic animals to produce animals that produce useful substances in eggs. There is a possibility to contribute. It also makes it possible to save damaged eggs such as precious wild birds and reptiles.

It is a figure explaining the preparation methods of the container produced in the Example of this invention. It is a figure explaining the preparation methods of the concave plastic film molded object produced in the Example of this invention. It is a figure explaining the heat-retaining method of a chicken shellless sperm egg performed in the Example of this invention.

  The egg to which the method of the present invention is applied is not particularly limited as long as it is a sperm egg having a shell, and examples thereof include eggs such as birds and reptiles. Bird eggs are preferred. Although chickens are used in the following examples, artificial hatching is considered to be easier when the egg size is smaller, and therefore, it is also easier for other birds such as quails with smaller egg sizes than chickens. It is considered applicable. In fact, it has succeeded in artificially hatching quail-shelled sperm eggs by the same method as described in the following examples. Some sperm eggs are commercially available such as chickens, and such commercially available edible eggs can also be used.

  In the method of the present invention, a transparent plastic film having oxygen permeability is used in order to hold a shellless sperm egg. Such a plastic film is not particularly limited, but a polymethylpentene film is preferable. The polymethylpentene film is marketed as a food storage wrap, has high oxygen permeability, and is suitable for vegetable storage. Since the polymethylpentene film for food preservation is commercially available (trade name “Fourap”), this commercially available polymethylpentene film for food preservation can be preferably used.

  In the method of the present invention, a sperm without a shell is held and kept warm in a recess of the plastic film formed by stretching the plastic film into a concave shape. Here, “stretch molding” means that the plastic film is plastically deformed by applying tension to the plastic film, and can be performed using human hands. This stretch molding can be performed by pressing a plastic film against a convex mold, locally stretching the plastic film, and plastically deforming the plastic film into a concave shape. Here, the sperm without a shell is held in the formed recess and hatched here, so that the size of the recess is preferably approximately the same as the size of the egg. Therefore, as the convex pressing mold, one supporting a shelled egg so that the longitudinal direction of the egg is in the vertical direction can be used. For example, as described in the following examples, a shelled egg is placed in the mouth of a milk bottle so that the longitudinal direction of the egg is in the vertical direction, and in this state, another film or cloth (in the following examples, stockings Fixed in this state by using a draining bag of a type), and the fixed shelled egg can be used as a stamping die. In addition, even if a shell egg is a raw egg, since the compressive strength of a longitudinal direction is large, even if it uses as a pressing die and a big pressure is applied, it does not break. However, the pressing die is not limited to an egg, and may be any one as long as a concave portion similar to the case of using an egg is formed. For example, it is commercially available for the head of a plastic bottle rocket. It is also possible to use a cushion with a slightly truncated tip. Cover the above-mentioned plastic film with the plastic film, hold the end of the plastic film with both hands and press strongly against the mold from above, the plastic film is stretched, plastically deformed, and corresponds to the convex mold A recess having a shape is formed. It is preferable to perform this process by two people and hold the four corners of a rectangular plastic film because the plastic film is uniformly and well stretched. As described above, when a shelled egg is used as a pressing mold, a recess having a shape and size corresponding to the shelled egg is formed. In addition, since the thickness of a film reduces by this stretch molding, it is thought that oxygen permeability further increases.

  It is important that there are no wrinkles in the recess that holds the shellless sperm egg. The present inventor has found that hatching becomes difficult when the concave film has wrinkles. Although it is possible to form a concave portion without wrinkles only by the above method using a pressing die, in order to ensure that there are no wrinkles, the concave portion of the concave portion is suspended in a state where a plastic film plastically deformed into a concave shape is suspended. It is preferable that the film is further pulled evenly by suction with an aspirator in the bottom direction. By this suction treatment, the film is uniformly pulled toward the bottom of the concave portion, and no wrinkle remains reliably.

  The concave plastic film molded body without wrinkles produced as described above is suspended in the container. Although it does not specifically limit as a container, The thing made from a transparent thermoplastic is preferable and the thing with a transparent lid | cover is preferable. As specifically described in the following examples, the concave plastic film molded body was formed by sandwiching the peripheral edge of the previously formed concave plastic film molded body between the lid of the container and the peripheral edge of the opening of the container. It can be easily suspended in the container. Of course, it is also possible to fix the peripheral part of the concave plastic film molded body to the peripheral part of the opening of the container with an adhesive, rubber band, adhesive tape or the like without using a lid. It is preferable to open at least one air hole in the container from the viewpoint of sufficient oxygen supply. In this case, it is preferable that the air hole is filled with a cotton plug in order to prevent contamination of germs through the air hole. Also, it is preferable that at least one, preferably about 6 to 12 air holes are formed in the plastic film in a portion above the portion in contact with the sperm egg.

  As described above, the plastic film molded body can be simply suspended in the container. However, the plastic film molded body is placed in the inverted frustoconical cage-shaped base that is suspended in the container. May be. Thereby, it is possible to prevent the concave portion of the plastic film molded body from hanging down due to the weight of the sperm egg or chick, but it is possible to hatch without using this pedestal. As such a basket-like pedestal, for example, a gardening article made of polypropylene or the like placed on the bottom of a flower pot can be used.

  Next, the hatching method of the present invention using the above instrument will be described. First, as a preparation stage, before cracking the shell, the shelled sperm egg is kept warm in an incubator. Insulation is a temperature suitable for each animal, and in the case of chickens, it is preferably 38 ° C. As for the heat retention period in the state with a shell, 2-3 days are preferable.

  Next, the sperm egg which kept warm is interrupted in the recessed part of the concave-shaped plastic film molded object suspended in the container produced as mentioned above. That is, the egg is broken in the manner of breaking a raw egg during cooking, and the contents are put into the recess. At this time, it is preferable to break the yolk at a stroke so as not to be damaged by the shell. For hatching, it is important to keep the unshelled sperm eggs in contact with liquid water and solid calcium salts. As the solid calcium salt, calcium lactate is preferable. In the case of chickens, the amount of calcium lactate is preferably about 0.2 to 0.35 g, and the amount of water is preferably about 2 to 3 mL. It is preferable that the calcium salt and the liquid water are placed in the concave portion of the concave plastic film molded body before the sperm egg is interrupted, and the sperm egg is interrupted thereon. If calcium salt and liquid water are initially placed in the recesses in this way, no further replenishment is necessary. In addition, when using calcium lactate powder, it is preferable that calcium lactate powder and pure water are gently added so that the calcium lactate does not dissolve in water and is not stirred or moved in water. Moreover, it is preferable to put an aqueous solution of a bactericide such as benzalkonium chloride at the bottom of the container in order to prevent contamination of bacteria and supply water.

  In this state, the container is placed in an incubator and kept warm. Insulation is a temperature suitable for each animal, and in the case of chickens, it is preferably 38 ° C. In order to prevent drying, the humidity in the incubator is preferably 100%. Further, the inside of the incubator is preferably sterilized in advance by spraying a bactericidal solution.

  It is preferable to roll the container while keeping the temperature in the incubator. Here, “rolling” means changing the direction of gravity applied to the embryo relative to the embryo. Rolling can be easily performed, for example, by tilting the shelf on which the container is placed about 5 to 15 degrees and changing the direction of the container from time to time. Although the frequency of rolling is not specifically limited, For example, it may be once to twice a day.

  By continuing the incubation in the above-mentioned state, the cleavage proceeds, the embryo develops into a chick, and finally hatches. Here, it is important to supply humidified oxygen into the container for at least the last 10%, preferably 10% to 25%, of the period from the start of incubation to hatching. In the case of chickens, hatching usually takes place from 18 to 21 days from the start of incubation, but preferably after the start of incubation (including the incubation period in a shelled state), humidified oxygen is continuously supplied until hatching. To do. The supply amount of the humidified oxygen is not particularly limited, but is usually about 300 mL / h to 1000 mL / h, preferably about 400 mL / h to 600 mL / h.

  During hatching, the chicks come out of the urinary membrane by themselves, but if the urine membrane cannot be broken by itself, it is preferable to incise the chorioallantoic membrane in the vicinity of the chicks. Thereby, the hatching rate can be increased.

  Hereinafter, examples of the present invention in which chicken shelled edible sperm eggs are artificially hatched will be described in detail.

1. Production of container (Fig. 1)
A commercially available styrene resin transparent plastic container (capacity 430 mL) 10 having a diameter decreasing from the opening toward the bottom was purchased. With the turbo lighter 12, a hole 14 having a diameter of about 1 cm was formed at a position about 1/3 from the bottom of the container 10 (1 in FIG. 1), and a cotton plug 16 was pushed into the hole 14 (2 in FIG. 1). On the other hand, the vicinity of the top of the cone of the conical cage-shaped polypropylene gardening article 18 having a flange at the opening and the surplus portion of the flange are cut with scissors to form an inverted truncated cone shape having a flange (FIG. 1). 3), suspended inside the container 10 to form a pedestal 18a (4 in FIG. 1). In addition, in the present Example, it performed about the case where this pedestal is used and the case where it is not used.

2. Production of Plastic Film Molded Body A commercially available polymethyltenpen film for food preservation (trade name Four Wrap, manufactured by Riken Technos) was purchased (1 in FIG. 2). On the other hand, the egg 21 with the shell is placed in the opening of the milk bottle 19 so that the longitudinal direction of the egg 21 is vertical, covered with a stocking-type draining bag 20, fixed with a rubber band 22, and the mold is It was produced (2 in FIG. 2). On the other hand, the polymethyl tempen film 17 is cut into a rectangular shape and covered from above so that the center part hits the pressing mold composed of the eggs 21, and the rectangular film is gripped with both hands and pushed downward to depress the pressing mold. The film was stretched by biting into the center and plastically deformed to form a recess 23a corresponding to the shape and size of the die. The film was pulled up vigorously and separated from the pressing die to obtain a concave plastic film molding 23.

  On the other hand, as shown in 5 of FIG. 2, a plastic funnel 26 is connected to the aspirator 24, a suction cup 28 having a hole at the bottom is pressed against the funnel 26, and the concave plastic film molded body 23 previously formed is pressed. Was attached to the opening of the suction cup 28 as shown in the figure, sucked by an aspirator, and the plastic film was further pulled in the direction of the bottom of the concave portion of the plastic film molded body 23 to stretch out the wrinkles completely.

3. Preparation of heat insulation of shellless sperm egg A commercially available edible sperm egg with a shell was purchased and stored at 15 ° C. in a refrigerator. Within the shelf life, the eggs were transferred to an incubator and kept at 38 ° C. for 55 hours.

  Put the benzalkonium chloride aqueous solution 30 (trade name 1000-fold diluted Osban solution) into the bottom of the container 10 that was prepared earlier to the extent that it does not touch the cotton plug, and then form a concave plastic film formed in the opening of the container 10 previously. The body 23 was covered (1 in FIG. 3). When the cage-shaped pedestal 18a (see 4 in FIG. 1) is used, the recess 23a is accommodated inside the cage-shaped pedestal 18a (the pedestal is not shown in FIG. 3). 0.25 g or 0.3 g of calcium lactate powder 31 and 2.5 mL or 3 mL of pure water 33 were placed on the bottom of the recess 23a. At this time, it was placed gently so that the calcium lactate powder 31 and the pure water 33 were not mixed.

  The shell of the edible sperm egg with the shell that had been kept warm was broken, and the contents were cut into the recess 23a. At this time, the shell was opened at a stretch so that the yolk was not damaged by the cracked shell (2 in FIG. 3). 6 to 8 air holes 32 were opened with a soldering iron in a region of the plastic film molded body 23 above the shellless sperm egg 36. The plastic film molding 23 was suspended by covering the transparent plastic lid 34 and sandwiching the peripheral portion of the plastic film molding 23 between the peripheral edge of the opening of the container (4 in FIG. 3). Excess film protruding outside the container 10 was cut off and removed.

4). Insulation of a shellless sperm egg The container constructed as described above, which holds a shellless sperm egg, was kept warm in an incubator at 38 ° C. and 100% humidity. Prior to the heat retention, an aqueous benzalkonium chloride solution was sprayed in the incubator to sterilize. The shelves in the incubator were inclined at an angle of 8 degrees and the side close to the door was down, and the shelves were rolled twice a day by changing the direction of the container. From the 16th day of the start of heat insulation (including heat insulation in a shelled state), using a commercially available oxygen gas cylinder with a regulator and an oxygen flow meter with a humidifier, oxygen was hatched in the container 10 at a flow rate of 500 mL / h with a catheter. Until continuously fed. This is done by connecting a gas pipe to an oxygen gas cylinder placed outside the incubator, and passing this gas pipe through the inside of the incubator through a hole provided in the incubator for passing the gas pipe etc. It was connected to the oxygen flow meter with a humidifier arranged in the above, and then the gas pipe was branched and connected to several catheters, and each catheter was inserted into the container from the air hole with a cotton plug of each container. Carefully observe the embryo from the 18th day of the start of incubation, where there is a possibility of hatching, and for chicks that cannot easily break the allantoic membrane on their own, the chorioallantoic membrane near the pupa is about 5 mm on the 18th or 19th day of the incubation. An incision was made to help the chicks break through the chorioallantoic membrane and come out.

5. Results When artificial hatching of shellless sperm eggs in 5 cases (3 cases without pedestal, 2 cases with pedestal) was performed by the method described above, the chicks hatched on the 20th day after the start of heat insulation (4 cases remaining). Died on the 13th day without starting the pedestal). Of the 4 cases that hatched, there were 1 case of chicks that broke through the allantoic membrane and 3 cases of incision of the chorioallantoic membrane. Three of the chicks that were born continue to be reared and observed (about 6 weeks after birth at the time of filing) and are growing normally.

DESCRIPTION OF SYMBOLS 10 Container 12 Turbolighter 14 Air hole 16 Cotton plug 17 Polymethyl tempen film 18a Base 18 Cone-shaped basket-shaped polypropylene gardening equipment 19 Milk bottle 20 Stocking-type draining bag 21 Egg 22 Rubber band 23 Concave plastic film molding 23a Recess 24 Aspirator 26 Funnel 28 Suction cup 30 Benzalkonium chloride aqueous solution 31 Calcium lactate powder 32 Air hole 33 Pure water 34 Lid 36 Shellless sperm egg

Claims (14)

A step of inserting a shelled sperm egg into a concave portion of a concave plastic film molded body without wrinkles suspended in a container, formed by stretching a transparent plastic film having oxygen permeability into a concave shape;
Placing the container in an incubator and keeping the interrupted shellless sperm egg in contact with liquid water and solid calcium salt; and
Supplying at least the last 10% of the period from the start of heat insulation to hatching, supplying humidified oxygen into the container,
Artificial hatching of shellless sperm eggs.   The method of claim 1, wherein the plastic film is a polymethylpentene film.   The method according to claim 1 or 2, wherein the calcium salt is calcium lactate.   The said calcium salt and liquid water are put into the recessed part of the said concave plastic film molded object, before interrupting the said sperm egg, and the said sperm egg is interrupted on this, The any one of Claims 1-3. The method described in 1.   The method according to claim 1, wherein the stretch molding includes pressing the plastic film against a convex mold to plastically deform the plastic film into a concave shape.   6. The method according to claim 5, wherein in a state where a plastic film plastically deformed into a concave shape is suspended, suction is made toward the bottom of the concave portion to ensure that there are no wrinkles in the concave portion.   The method according to any one of claims 1 to 6, wherein the temperature is kept in a state where an aqueous bactericide solution is placed at the bottom of the container.   The method according to claim 1, wherein the container has at least one air hole.   The method according to any one of claims 1 to 8, wherein the plastic film has at least one air hole in a portion above a portion in contact with the sperm egg.   The method according to claim 1, wherein the plastic film molded body is disposed in an inverted truncated cone-shaped pedestal suspended in the container.   The method of any one of Claims 1-10 which further includes the process of rolling a container during heat retention.   The method according to any one of claims 1 to 11, further comprising a step of incising the chorioallantoic membrane in the vicinity of the chick's pupae when the chick can't break the chick's own membrane at the time of hatching.   The method according to claim 1, wherein the sperm egg is an avian sperm egg.   The method of claim 13, wherein the bird is a chicken.

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